摘要：

Background: Prediction P450-mediated metabolism in humans constitutes an essential part of drug development. A variety of recombinant expression systems have been employed, however only on a small scale, to predict P450 -mediated metabolism in man. Hypothesis: We argue that bacterial are ideally suited for semipreparative biooxidations, as they can be grown to high density and are technically not challenging. Methods: Human P450s were expressed in E. coli using published procedures. For biofermentations, E. coli were grown, if required in up to 100 l biofermenters, or employing BioWave bioreactors (Wave Biotech AG, Switzerland). Results: We demonstrated that the catalytic properties of several P450 isoforms when expressed in E. coli faithfully mimicked metabolic pathways of xenobiotics in human liver. The technical applicability of these systems for biofermentation is evidenced by the industrial applications presented here. These were conducted by two pharmaceutical companies, involving structurally diverse compounds, and utilized different bioreactor techniques. The strategies employed, resulted in highly efficient, semipreparative biooxidations of drugs and drug candidates. For example CYP3A4-mediated biooxidation of AAG561, a new drug compound in development by Novartis against anxiety and depression, revealed four metabolites in quantities of up to 13. 6 mg (1 litre culture). In another application, using optimized conditions, metabolism of amodiaquine was carried out in 5 x 2L flasks each containing 80 nmol of CYP2C8. Metabolites were isolated by flash chromatography and 172 mg of desethyl-amodiaquine in > 99% purity was recovered. Conclusions: The results presented demonstrate that E. coli expressing human P450s are ideally suited for semi-preparative production of drug metabolites for structural and drug safety studies, as will be increasingly required by regulators.

展开